Kangchu Li

HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells

Hypoxia inducible factor 1 (HIF-1), the key mediator of hypoxia signaling pathways, has been shown involved in hypoxia-induced radioresistance. However, the underlying mechanisms are unclear. The present study demonstrated that both hypoxia and hypoxia mimetic cobalt chloride could increase the radioresistance of human cervical cancer Hela cells. Meanwhile, ectopic expression of HIF-1 could enhance the resistance of Hela cells to radiation, whereas knocking-down of HIF-1 could increase the sensitivity of Hela cells to radiation in the presence of hypoxia. N-Myc downstream-regulated gene 2 (NDRG2), a new HIF-1 target gene identified in our lab, was found to be upregulated by hypoxia and radiation in a HIF-1-dependent manner. Overexpression of NDRG2 resulted in decreased sensitivity of Hela cells to radiation while silencing NDRG2 led to radiosensitization. Moreover, NDRG2 was proved to protect Hela cells from radiation-induced apoptosis and abolish radiation-induced upregulation of Bax. Taken together, these data suggest that both HIF-1 and NDRG2 contribute to hypoxia-induced tumor radioresistance and that NDRG2 acts downstream of HIF-1 to promote radioresistance through suppressing radiation-induced Bax expression. It would be meaningful to further explore the clinical application potential of HIF-1 and NDRG2 blockade as radiosensitizer for tumor therapy.

The role of protein kinase C in the opening of blood-brain barrier induced by electromagnetic pulse.

The aim of this study was to determine the role of protein kinase C signaling in electromagnetic pulse (EMP)-induced blood-brain barrier (BBB) permeability change in rats. The protein level of total PKC and two PKC isoforms (PKC-alpha, and PKC-beta II) were determined in brain cerebral cortex microvessels by Western blot after exposing rats to EMP at 200kV/m for 200 pulses with 1Hz repetition rate. It was found that the protein level of PKC and PKC-betaII (but not PKC-alpha) in cerebral cortex microvessels increased significantly at 0.5h and 1h after EMP exposure compared with sham-exposed animals and then recovered at 3h. A specific PKC antagonist (H7) almost blocked EMP-induced BBB permeability change. EMP-induced BBB tight junction protein ZO-1 translocation was also inhibited. Our data indicated that PKC signaling was involved in EMP-induced BBB permeability change and ZO-1 translocation in rat.

EMP-induced alterations of tight junction protein expression and disruption of the blood-brain barrier.

The blood-brain barrier (BBB) is critical to maintain cerebral homeostasis. In this study, we examined the effects of exposure to electromagnetic pulse (EMP) on the functional integrity of BBB and, on the localization and expression of tight junction (TJ) proteins (occludin and ZO-1) in rats. Animals were sham or whole-body exposed to EMP at 200 kV/m for 400 pulses. The permeability of BBB in rat cerebral cortex was examined by using Evans Blue (EB) and lanthanum nitrate as vascular tracers. The localization and expression of TJ proteins were assessed by western blot and immunofluorescence analysis, respectively. The data indicated that EMP exposure caused: (i) increased permeability of BBB, and (ii) altered localization as well as decreased levels of TJ protein ZO-1. These results suggested that the alteration of ZO-1 may play an important role in the disruption of tight junctions, which may lead to dysfunction of BBB after EMP exposure.

Comparative Investigations on the Protective Effects of Rhodioside, Ciwujianoside-B and Astragaloside IV on Radiation Injuries of the Hematopoietic System in Mice

The aim of this study was to investigate the protective effects of three glycosides (rhodioside, ciwujianoside‐B and astragaloside IV) on the hematopoietic system in the mice exposed to γ‐rays, and to examine the possible mechanisms involved. Mice were pretreated with the glycosides (40 mg/kg, i.g.) daily for 7 days prior to radiation. The survival of mice pretreated with three glycosides after total body irradiation (6.0 Gy) was examined. Peripheral blood leucocytes and endogenous spleen colony counts, colony‐forming unit‐granulocyte macrophage assay, analysis of DNA content and apoptosis rate determination were performed to evaluate the effects of the three glycosides on hematogenesis. The fragmentation of double‐stranded DNA in lymphocytes was detected by the comet assay. The changes in cell cycle were analysed by flow cytometry. Furthermore, the expression levels of Bcl‐2, Bax and nuclear factor‐kappa B (NF‐κB) were measured by western blot and the electrophoretic mobility shift assay. The results showed that pretreatment with all of the glycosides improved survival time and increased the number of leucocytes, spleen colonies and granulocyte‐macrophage colonies in mice exposed to 6.0 Gy γ‐radiation. Rhodioside showed more protective efficacy than both ciwujianoside‐B and astragaloside IV. All three glycosides significantly increased the proliferation abilities of bone marrow cells, and decreased the ratio of cells in G0/G1 phase. Further analysis showed that these three glycosides were able to decrease DNA damage and the increment in the Bax/Bcl‐2 ratio induced by radiation. In summary, the three glycosides showed radioprotective effects on the hematopoietic system in mice, which was associated with changes in the cell cycle, a reduction in DNA damage, and down‐regulation of the ratio of Bax/Bcl‐2 in bone marrow cells exposed to radiation. Copyright

Knock-down of NDRG2 sensitizes cervical cancer Hela cells to cisplatin through suppressing Bcl-2 expression

BackgroundNDRG2, a member of N-Myc downstream regulated gene family, plays some roles in cellular stress, cell differentiation and tumor suppression. We have found that NDRG2 expression in cervical cancer Hela cells increases significantly upon stimulation with cisplatin, the most popular chemotherapeutic agent currently used for the treatment of advanced cervical cancer. This interesting phenomenon drove us to evaluate the role of NDRG2 in chemosensitivity of Hela cells.MethodsIn the present study, RNA interference was employed to down-regulate NDRG2 expression in Hela cells. RT-PCR and Western blot were used to detect expression of NDRG2, Bcl-2 and Bax in cancer cells. Real-time PCR was applied to detect miR-15b and miR-16 expression levels. Drug sensitivity was determined with MTT assay. Cell cloning efficiency was evaluated by Colony-forming assay. Apoptotic cells were detected with annexin V staining and flow cytometry.ResultsIn vitro drug sensitivity assay revealed that suppression of NDRG2 could sensitize Hela cells to cisplatin. Down-regulation of NDRG2 didn’t influence the colony-forming ability but promoted cisplatin-induced apoptosis of Hela cells. Inhibition of NDRG2 in Hela cells was accompanied by decreased Bcl-2 protein level. However, Bcl-2 mRNA level was not changed in Hela cells with down-regulation of NDRG2. Further study indicated that miR-15b and miR-16, two microRNAs targetting Bcl-2, were significantly up-regulated in NDRG2-suppressed Hela cells.ConclusionsThese data suggested that down-regulation of NDRG2 could enhance sensitivity of Hela cells to cisplatin through inhibiting Bcl-2 protein expression, which might be mediated by up-regulating miR-15b and miR-16.

Effects of PEMF exposure at different pulses on osteogenesis of MC3T3-E1 cells

OBJECTIVE Pulsed electromagnetic fields (PEMFs) were considered to be a factor which may affect osteogenesis of osteoblasts, but the effects were diverse with different PEMF parameters. The aim of the current study is to explore the effects of exposure to PEMFs at different pulse number on osteogenesis of osteoblasts. DESIGN The mouse osteoblast-like MC3T3-E1 cells were exposed to 0, 400 or 2800 pulses 400kV/m PEMF and the proliferation, differentiation and mineralization of cells were observed after PEMF exposure by the methods of MTT, biochemical measurement, real-time PCR and Alizarin Red assay. RESULTS Compared with 0 pulses groups, the growth curve, alkaline phosphatase (ALP) activity, mRNA level of osteocalcin (OCN) and mineralized nodule formation of MC3T3-E1 cells did not change after 400 pulses PEMF exposure, but decreased after 2800 pulses PEMF exposure. It suggested that under our experimental conditions, only 2800 pulses 400kV/m PEMF exposure can suppress the proliferation, differentiation and mineralization of MC3T3-E1 cells, but 400 pulses 400kV/m PEMF exposure cannot. CONCLUSIONS Pulse number is another involved parameter which may influence the effects of PEMF on osteogenesis of osteoblasts.

Inhibition of Bone Formation by High Intensity Pulsed Electromagnetic Field in Mc3t3-E1 Cells

To investigate the efiects of pulsed electromagnetic fleld (PEMF) with high electric fleld intensity on bone formation in murine osteoblast-like MC3T3-E1 cells, proliferation, alkaline phosphotase (ALP) activity, mineralized nodule formation, Collagen Type I (COL-I) and core-binding factor (Cbf)a1 mRNA expression, and bone morphogenetic protein (BMP)2/4 and mothers against decapentaplegic (Smad)1/5/8 protein expression were examined in cultured MC3T3-E1 cells after exposure to PEMF at the fleld intensities of 0kV/m, 50kV/m or 400kV/m for 400 consecutive pulses daily for 7 consecutive days. After 50kV/m of PEMF exposure, none of the above parameters of MC3T3-E1 cells changed signiflcantly when compared to the control groups. However, the proliferation, ALP activity and mineralized nodule formation of MC3T3-E1 cells in 400kV/m PEMF exposure groups decreased signiflcantly although COL-I and Cbfa1 mRNA expression and BMP2/4 and Smad1/5/8 protein expression did not change. The PEMF we used at high electric fleld intensity suppressed proliferation, difierentiation and

Effects of long-term 50Hz power-line frequency electromagnetic field on cell behavior in Balb/c 3T3 cells.

Power-line frequency electromagnetic field (PF-EMF) was reported as a human carcinogen by some epidemiological research, but the conclusion is lack of robust experiment evidence. To identify the effects of long-term PF-EMF exposure on cell behavior, Balb/c 3T3 cells in exponential growth phase were exposed or sham-exposed to 50 Hertz (Hz) PF-EMF at 2.3 mT for 2 hours (h) one day, 5 days every week. After 11 weeks exposure, cells were collected instantly. Cell morphology was observed under invert microscope and Giemsa staining, cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and apoptosis was examined by flow cytometry, the protein level of Proliferating Cell Nuclear Antigen (PCNA) and CyclinD1 was detected by western blot, cell transformation was examined by soft agar clone assay and plate clone forming test, and cell migration ability was observed by scratch adhesion test. It was found that after PF-EMF exposure, cell morphology, apoptosis, cell migration ability and cell transformation didn’t change. However, compared with sham group, cell viability obviously decreased and cell cycle distribution also changed after 11 weeks PF-EMF exposure. Meanwhile, the protein level of PCNA and CyclinD1 significantly decreased after PF-EMF exposure. These data suggested that although long-term 50Hz PF-EMF exposure under this experimental condition had no effects on apoptosis, cell migration ability and cell transformation, it could affect cell proliferation and cell cycle by down-regulation the expression of PCNA and CyclinD1 protein.

Effects of Pulsed Electromagnetic Field on Differentiation of HUES-17 Human Embryonic Stem Cell Line

Electromagnetic fields are considered to potentially affect embryonic development, but the mechanism is still unknown. In this study, human embryonic stem cell (hESC) line HUES-17 was applied to explore the mechanism of exposure on embryonic development to pulsed electromagnetic field (PEMF) for 400 pulses at different electric field intensities and the differentiation of HUES-17 cells was observed after PEMF exposure. The expression of alkaline phosphatase (AP), stage-specific embryonic antigen-3 (SSEA-3), SSEA-4 and the mRNA level and protein level of Oct4, Sox2 and Nanog in HUES-17 cells remained unchanged after PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m. Four hundred pulses PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m did not affect the differentiation of HUES-17 cells. The reason why electromagnetic fields affect embryonic development may be due to other mechanisms rather than affecting the differentiation of embryonic stem cells.

EMP-induced BBB-disruption enhances drug delivery to glioma and increases treatment efficacy in rats: EMP Enhances Drug Delivery to Glioma

Chemotherapy on gliomas is not satisfactorily efficient because the presence of blood-brain barriers (BBB) leads to inadequate exposure of tumor cells to administered drugs. In order to facilitate chemotherapeutics to penetrate BBB and increase the treatment efficacy of gliomas, electromagnetic pulse (EMP) was applied and the 1-(2-Chlorethyl)-cyclohexyl-nitrosourea (CCNU) lomustine concentration in tumor tissue, tumor size, tumor apoptosis, and side effects were measured in glioma-bearing rat model. The results showed that EMP exposure could enhance the delivery of CCNU to tumor tissue, facilitate tumor apoptosis, and inhibit tumor growth without obvious side effects. The data indicated that EMP-induced BBB disruption could enhance delivery of CCNU to glioblastoma multiforme and increase treatment efficacy in glioma-bearing rats. Bioelectromagnetics. 39:60-67, 2018.